Within this application several publications are referenced by arabic numbers within parentheses. Full citations for these publications may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which the invention pertains.
The plasmids pHRT25 and pHRTRX2 were deposited on Oct. 25, 1988 pursuant to, and in satisfaction of, the requirements of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852 under ATCC Accession Nos. 67117 and 67828, respectively.
Acquired Immune Deficiency Syndrome (AIDS) is an epidemic characterized by a marked depletion of the cellular immune response. The causative agent of the disease is now firmly established to be the human retrovirus known as Human Immunodeficiency virus (HIV), which has various forms (1-4,32,33). Efforts to arrest the spread of this virus are being made on two broad fronts: the development of antiviral vaccines which might allow immunized individuals to resist infection, and the development of antiviral drugs which would specifically retard or arrest viral replication. There have been many efforts to identify antiviral drugs with inhibitory activity against viral enzymes. One prominent target of such drugs is the virion-associated enzyme reverse transcriptase (5-8), required for the synthesis of the proviral DNA soon after infection (10,34,35). Indeed, the major antiviral drug currently prescribed, 3'-azido-3'-deoxythymidine (AZT), acts, after conversion to the corresponding triphosphate, as an inhibitor of the reverse transcriptase enzyme (36).
Several disclosures in the art concern the production of a polypeptide having reverse transcriptase activity by bacteria transformed with genetically engineered vectors. One disclosure involves the shotgun cloning into Escherichia coli of total genomic DNA isolated from the cells of warm-blooded vertebrate animals, e.g. fowl liver cells [Japanese patent publication no. 56087600].
Two other disclosures detail the expression of a segment of the retrovirus pol gene to produce reverse transcriptase in E. coli in high yield (15,16).
Co-pending, co-assigned U.S. patent application Ser. No. 731,128, filed May 6, 1985 describes the expression of enzymatically active reverse transcriptase from Moloney murine leukemia virus (MuLV) in relatively high yield.
Co-pending, co-assigned U.S. patent application Ser. No. 149,703, filed Jan. 29, 1988 describes the expression of polypeptides having DNA polymerase activity and substantially no ribonuclease activity. Much of the disclosure of U.S. application Ser. No. 149,703 has been published (31).
The expression of HIV reverse transcriptase in bacterial systems (26,27,28,29) and in yeast (30) has been reported.
The subject application is a continuation-in-part of co-pending, co-assigned U.S. patent application Ser. No. 865,156, filed May 20, 1986. Much of the disclosure of U.S. application Ser. No. 865,156 has been published (20). In U.S. Ser. No. 865,156, applicants described the expression of polypeptides having reverse transcriptase activity. Applicants' present invention provides for a series of plasmids derived from the parent construct (disclosed in U.S. Serial No. 865,156) by removal of unnecessary portions of the pol gene. These plasmids unexpectedly result in the formation of proteins with increased stability and activity.